XMRV and Culturing, HERV's and more

I know what you're saying UKXMRV but I question - as you noted - whether its feasible. We all have CFS and I imagine it takes Kurt deal of time and energy to put these posts together. I would note that much of what we post on these forums is opinion. I do recognize that because Kurt is posting some kind of hard hitting stuff that you want to be sure to separate the two - but you may asking too much.

Thanks for getting that stuff on HERV's Gerwyn!

The genotyping was done during the Science study -when they were able to find XMRV without using culture or activating cells - possibly a different situation than we have now. I did note that the WPI is continuing to sequence the XMRV they find; this is a check on their original findings and will tell us whether its a HERV or something else. One would think they must know by now- and again we're not seeing any retractions or any sense that things are falling apart.

Adam I agree that the HERV argument is pure speculation. The important part of that blog for me is not whether HERV's are present or not but why the shift occurred from not needing to activate the cells to find XMRV to needing to activate the cells in order to find XMRV. If I got it right and I assume I did - Gerwyn would have corrected me by now - something rather dramatic changed. Why did the virus get so much harder to find all of a sudden? It puts Dr. Mikovits lambasting the other research groups for not culturing the virus - when there was no evidence I could find - of the WPI doing that - in a rather strange light. Maybe she communicated with them after the paper came out. In fact she told me that she told the Imperial Group how difficult it was to find the virus...so they were warned.

Kurts theory is one to explain that strange situation.

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i,m sorry you cant find the evidence but it is clearly in the paper there is no shift nothing changed

so yes I am correcting you

Dr M might have assumed that other virologists would actually read the paper and felt it unnessary to state the absolutely obvious. ALL latent phase viruses are incredibly hard to detect especially iof you dont look in the right place

they did two lots of pcr i will post the details shortly and i will contextualise the other studies at the same time so that lay people can see the very plain differences

Much of what has been discussed on these threads is speculation, whether it be on cohorts, assay techniques, researchers motivations, whatever, perhaps informed speculation, perhaps not. I'm not in a position to tell. In fact without speculation, these threads would have ended between the last published XMRV article and the next one to come.

As ably outlined by Cort, the picture on XMRV is pretty mixed at present. Some issues remain unexplained and all we are doing is trying to fill in the gaps in the absence of more solid information. Personally I'm happy that Kurt, or anyone else, is offering up alternative interpretations of the findings. If XMRV doesn't turn out to be the culprit, or what they found wasn't XMRV, then I'd like to think there are alternatives to going back to Wessley/Reevesville.

Kurt has been asked to indicate what is fact (sic) and what is speculation and or opinion, and to provide references. Why single Kurt out?

The dialogue here has largely been between Kurt and Gerwyn. To be perfectly honest most of Gerwyn's contributions are like alphabet soup to me. I don't have the background and, while I might understand the science in time, frankly I don't have the inclination as I don't feel qualified to offer an opinion. Kurt has described his background and its limitations. As far as I'm aware, Gerwyn has self described himself in correspondence as a psychologist and has stated on this thread that he has educated himself on virology. I have to ask if this makes Gerwyn any more qualified to speak on the subject than anyone else, in fact he could be a complete nutter. As could we all.

As for references, this was Gerwyn's response to the first post on the Interactions between APOBEC3 proteins, HIV-1, and XMRV at NIH on April 1st thread :

“cell cell transfer is another way of doing it”

What is one to make of that? Is this speculation, opinion, where's the reference? I'm sure he's right but how can I tell?

PLEASE Gerwyn, this is not a personal attack on you, your expertise or your integrity. But you must appreciate that I take any statement made on this forum, that does not originate from a relevant professional, with a little pinch of salt.

Either require anyone posting here to specify fact, opinion, rumour etc and fully reference any statement claimed to be fact, or don't. Personally I believe it would stifle many interesting discussions.

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It is a shame that i am also a qualified microbiologist as well then is it not---i dont think anyone else on this forum is . If you genuinely cant understand the concept of cell cell transfer I am sorry .I could not think of a simpler way of putting it. It means moving between a cell and another cell without need to go into the blood. I hope the soup is a little clearer now

I have never stated that I have educated myself in Virology You must be confusing me with someone else

You must pretty full of salt i would imagine!

I am a little confused .Are you saying that you take Kurts comments as emmanating from a relevant professional? I think that he mentioned he was self taught in Biology

You are quite right to be wary of random nutters on forums though it happens a lot

I appreciate Kurt and Gerwyn's post. Alot of the stuff goes right over my unscientific head....but they are really digging into the details of these studies and this disease.

When their posts first started I was "on gerwyn's side" and felt pissy toward Kurt...why is he being such a downer..but the longer the uncertainty and unclarity remains around xmrv, the more it is in MY best interest to hear all sides of the story.

Much of the "hearsay" Kurt talks about in his posts I have heard from several of my cfid's docs too. I have also heard the the PRO XMRV side from other docs. Yes, I see a lot of doc's...I'm working hard to get well. So there are many differing views out there still...and many unanswered questions.

When i read gerwyn and kurt's posts they just confirm what i am hearing from my outside sources of cfid's info....

i hope kurt has a thick skin and is not offended by some of the recent posts here...and keeps posting his viewpoint....i thought the post criticizing his tone was unnecessary and disrespectful...i don't notice a tone from kurt...just lots of hardwork and a presentation of an alternate view.

and i hope gerwyn keeps posting his smart analysis too (strangely from gerwyn's user name i thought he was a she?!?!)

Ok, totally out of my depth - totally - but, along with letting us know he was a microbiologist, didn't Gerwyn recently (last day or two) also explain some key issues and terms that clear up this culturing business? Was there not an issue with the different meanings of "amplification" for instance?

I'm going on memory here, never a good idea with me, but I'm pretty sure I read all of this very recently and that it all made a great deal of sense.

I don't think that Gerwyn or Dr. Yes have responded to the my understanding that, based on a reading of the Science document, the WPI didn't activate or culture the cells for the PCR tests for the Science paper. My reading suggests that they were able to find the virus without culturing at that point.

Now, however, they state that culturing is absolutely needed and that any studies that do not culture will not find it.

That brings up two possibilities that I, a laymen! can see - the virus was found in much smaller quantities in the second group they tested (ie Dr. Vernon is on the mark with her concerns about how untypical that first group might be). This would be a nice solution - XMRV is there but its more difficult to find than expected.

I think there are problems with this solution; I just don't think researchers are stupid and I don't think they like to be proved wrong; I think the first studies tried to find the virus and couldn't. They all showed that they could detect XMRV in low levels and yet couldn't find it in their samples. I know there are lots of factors involved; the primary one, though, seemed to be the lack of activation - so when it became clear that the WPI didn't activate their cells either - this made me think this factor is not so important. I know there are more questions about the first two validation studies but it removed some doubts about them for me.

It could still be that XMRV is present in the blood of very ill patients but not in the blood of less patients. This is apparently Dr. Vernon asked the WPI to provide more info on those patients; she feels that the hunt for XMRV is going to stop unless they get some positive results.

or as Kurt pointed out:

The culturing process itself is bringing something out in the CFS blood samples (but not the controls) that looks very much like XMRV but is not actually the bug.

In any case, I have a feeling this will all be moot soon as more studies pour out.

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i will answer the questions about the pcr so everyone can see

The comments about the culture and looking like the bug but not xmrv -priceless!

I don't think that Gerwyn or Dr. Yes have responded to the my understanding that, based on a reading of the Science document, the WPI didn't activate or culture the cells for the PCR tests for the Science paper. My reading suggests that they were able to find the virus without culturing at that point.

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It was my understanding that they DID activate (and culture) some cells prior to PCR, but not others. Certain subsets of cells were cultured with IL-2, others were not.. and then PCR was done. That's how I read the supplemental section anyway. Haven't figured out why they did it that way, but as I see above Gerwyn is planning to enlighten us on this subject. That should be entertaining.

Sorry I haven't been able to respond much, my brain really is deep-fried lately, and I have lost the ability to sift through specialist methodologies (or probably even a Julia Child cookbook) of late. Besides, at this point we really are all speculating about the future; even if a seasoned retrovirologist was a forum member and we understood all the nuances of the studies perfectly, we would still have no indication of what will be coming out later this year.

Thanks - here the sections that I thought were relevant are again. (Sorry to do this again Dannybex - look away!! ))

DNA and RNA isolation. Whole blood was drawn from subjects by venipuncture using standardized phlebotomy procedures into 8-mL greencapped Vacutainers containing the anti-coagulant sodium heparin (Becton Dickinson, Franklin Lakes, NJ). Plasma was collected by centrifugation, aspirated and stored at -80 C for later use. The plasma was replaced with PBS and the blood resuspended and further diluted with an equal volume of PBS. PBMC were isolated by layering the diluted blood onto Ficoll-Paque PLUS (GE Healthcare, Waukesha, WI), centrifuging for 22 min at 800 g, aspirating the PBMC layer and washing it once in PBS. The PBMC (approximately 2 x 10 cells) were centrifuged at 500x g for 7 min and either stored as unactivated cells in 90% FBS and 10% DMSO at -80 C for further culture and analysis or resuspended in TRIzol (Invitrogen, Carlsbad, CA) and stored at -80 C for DNA and RNA extraction and analysis.DNA was isolated from TRIzol preps according the to manufacturer’s protocol and also isolated from frozen PBMC pellets using the QIAamp DNA Mini purification kit (QIAGEN, Valencia, CA) according to the manufacturer’s protocol, and the final DNA was resuspended in RNase/DNasefree water and quantified using the Quant-iT Pico Green dsDNA Kit (Invitrogen, Carlsbad, CA). RNA was isolated from TRIzol preps according to the manufacturer’s protocol and quantified using the Quant-iT Ribo Green RNA kit (Invitrogen, Carlsbad, CA). cDNA was made from RNA using the iScript Select cDNA synthesis kit (Bio-Rad, Hercules, CA) according to the manufacturer’s protocol.

PCR. To avoid potential problems with laboratory DNA contamination, nested PCR was performed with separate reagents in a separate laboratory room designated to be free of high copy amplicon or plasmid DNA. Negative controls in the absence of added DNA were included in every experiment. Identification
of XMRV gag and env genes was performed by PCR in separate reactions. Reactions were performed as follows: 100 to 250 ng DNA, 2 L of 25 mM MgCl2, 25 L of HotStart-IT FideliTaq Master Mix (USB Corporation, Cleveland, OH), 0.75 L of each of 20 M forward and reverse oligonucleotide primers in reaction volumes of 50 L. For identification of gag, 419F (5’ATCAGTTAACCTACCCGAGTCGGAC-3’) and 1154R (5’GCCGCCTCTTCTTCATTGTTCTC-3’) were used as forward and reverse primers. For env, 5922F (5’- GCTAATGCTACCTCCCTCCTGG-3’) and 6273R (5’-GGAGCCCACTGAGGAATCAAAACAGG-3’) were used. For both gag and env PCR, 94C for 4 min initial denaturation was performed for every reaction followed by 94C for 30 seconds, 57C for 30 seconds and 72C for 1 minute. The cycle was repeated 45 times followed by final extension at 72C for 2 minutes. Six microliters of each reaction product was loaded onto 2% agarose gels in TBE buffer with 1 kb+ DNA ladder (Invitrogen, Carlsbad, CA) as markers. PCR products were purified using Wizard SV Gel and PCR Clean-Up kit (Promega, Madison, WI) and sequenced. PCR amplification for sequencing full-length XMRV genomes was performed on DNA amplified by nested or semi-nested PCR from overlapping regions from PBMC DNA. For 5’ end amplification of R-U5 region, 4F (5’CCAGTCATCCGATAGACTGAGTCGC-3’) and 1154R was used for first round and 4F and 770R (5’-TACCATCCTGAGGCCATCCTACATTG-3’) was used for second round. For regions including gag-pro and partial pol, 350F(5’GAGTTCGTATTCCCGGCCGCAGC-3’) and 5135R (5’- CCTGCGGCATTCCAAATCTCG-3’) was used for first round followed by second round with 419F and 4789R (5’-GGGTGAGTCTGTGTAGGGAGTCTAA-3’). For regions including partial pol and env region, 4166F (5’- CAAGAAGGACAACGGAGAGCTGGAG-3’) and 7622R (5’GGCCTGCACTACCGAAAT TCTGTC-3’) were used for first round followed by 4672F (5’GAGCCACCTACAATCAGACAAAAGGAT-3’) and 7590R (5’- CTGGACCAAGCGGTTGAGAATACAG-3’) for second round. For the 3’ end including the U3-R region, 7472F (5’-TCAGGACAAGGGTGGTTTGAG-3’) and 8182R (5’-CAAACAGCAAAAGGCTTTATTGG-3’) were used for first round followed by 7472F and 8147R (5’-CCGGGCGACTCAGTCTATC-3’) for second round. The reaction mixtures and conditions were as described above except for the following: For larger fragments, the final extension was done at 68C for 10 min instead of 72C for 2 min. All second round PCR products were column purified as described above and overlapping sequences were determined with internal primers. oNested RT-PCR for gag sequences was done as described (5) with modifications. GAG-O-R primer was used for 1st strand synthesis; cycle conditions were 52oC annealing, for 35 cycles. For second round PCR, annealing was at 54C for 35 cycles. PCR analysis performed on 20 of the identical patient PBMC DNA specimens stored at the NCI (Frederick, MD) since 2007confirmed nearly identical gag sequences, thereby diminishing the possibility of laboratory contamination as a source of XMRV.

It was my understanding that they DID activate (and culture) some cells prior to PCR, but not others. Certain subsets of cells were cultured with IL-2, others were not.. and then PCR was done. That's how I read the supplemental section anyway. Haven't figured out why they did it that way, but as I see above Gerwyn is planning to enlighten us on this subject. That should be entertaining.

Sorry I haven't been able to respond much, my brain really is deep-fried lately, and I have lost the ability to sift through specialist methodologies (or probably even a Julia Child cookbook) of late. Besides, at this point we really are all speculating about the future; even if a seasoned retrovirologist was a forum member and we understood all the nuances of the studies perfectly, we would still have no indication of what will be coming out later this year.

The next time primary cells are mentioned is here in the Viral transmission section:

Viral transmission. Frozen cell-free plasma and 0.22 m filtered cell free supernatants from PBMC and T cell cultures were diluted 1:1 with tissue culture media and 600 L aliquots were added to a six-well culture plate with the LNCaP
ocell line (50% confluent) or a million primary activated CD4+ T cells isolated from healthy donors. The plates were centrifuged for 5 min at 1500 RPM, rotated 1806 and centrifuged again for 5 min. The entire cycle was repeated once and cells were then diluted in their growth media. For cell-cell transmission, 1 x 10oT cells or PBMC without any IL-2 in the growth media were added to a six-well culture plate with the LNCaP cell line (50% confluent) in 1 mL of media for 3 h. After 1 hr, T cells in suspension were removed and the LNCaP cells were grown for several passages in the absence of IL-2 which caused any remaining T cells to die. At the times after transmission indicated, protein analysis was done by western blot and flow cytometry. Genotyping. The rs486907 R462Q SNP was genotyped using Applied Biosystems’ Taqman 5' nucleotidase assays, Taqman Universal PCR Master Mix: No AmpErase UNG, and 5 ng of genomic DNA. The thermal cycling conditions consisted of an initial hold at 95o C for 10 minutes followed by 50 cycles of a 15 second 95C denaturation step and a one minute 60o C annealing and extension step. A 7900HT instrument was used to detect fluorescent probes, and Sequence Detection Software (SDS) v2.2 was used to discriminate alleles and call genotypes (Applied Biosystems, Foster City, CA). The variant is in Hardy-Weinberg equilibrium in both cases and controls. A Chi square test was performed for both genotypes and alleles of RNASEL comparing XMRV negative and XMRV positive controls. Both tests were not significant and the allele test is displayed in Table S2. Homozygous R462Q variant of RNASEL is represented in approximately 13% of the human population (S6, S7).

Its really not clear to me. The activation section is below the PCR section. Activated cells are not noted in connection with the PCR yet they are in other sections. Why point out that cells are activated on one section and not another? Did they use the primary cells for the PCR? Or did they just used the PBMC's as stated? What are primary cells? Does that denote something in particular?

They used PBMC's for the PCR; in the activation section they talk about splitting up the cells into their types - but they didn't do that for the PCR; they simply refer to PBMC's.

Note that it says the cells were either stored as unactivated cells for further culture or resuspended in Trizol for DNA and RNA extraction; ie one set of cells was cultured; the other set of cells was not - the uncultured cells were used for DNA and RNA extraction. I don't know how else to read that.

The PBMC (approximately 2 x 10 cells) were centrifuged at 500x g for 7 min and either stored as unactivated cells in 90% FBS and 10% DMSO at -80 C for further culture and analysis or resuspended in TRIzol (Invitrogen, Carlsbad, CA) and stored at -80 C for DNA and RNA extraction and analysis

Ok, totally out of my depth - totally - but, along with letting us know he was a microbiologist, didn't Gerwyn recently (last day or two) also explain some key issues and terms that clear up this culturing business? Was there not an issue with the different meanings of "amplification" for instance?

I'm going on memory here, never a good idea with me, but I'm pretty sure I read all of this very recently and that it all made a great deal of sense.

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Yes, it seems to me that Gerwyn or someone else has patiently explained or countered every item, then someone has doggedly circled round the block and brought it up again and again. I don't know if that means they didn't understand the explanation, or they just didn't like the inference.

And FWIW, I don't need anyone to keep me from getting "too hopeful" (whatever that is) or to keep my feet on the ground. No need to save me from myself. I feel insulted and talked down to if anyone is posting negative speculation on XMRV just to keep me from getting too excited about it. Like Adam, the rumors and speculations I've seen here have been predominately negative on XMRV. Of course, if there is some real science that disproves WPIs preliminary study, bring it on. None has appeared so far.

And to describe any of Dr Mikovits's statements, at least those I'm aware of, as "lamblasting" anyone is hyperbole itself. Dr Vernon's speculation, if what Cort says of her is true, that purported flaws in WPIs study might somehow lower the interest of the rest of the scientific community in XMRV just isn't supportable by the facts. There are studies on XMRV going on all over the world. Governments, private researchers and BigPharma are all interested, no matter whether the criticisms of WPI are born out or proven to be more smoke.

What I am seeing is that whenever someone expresses a hopeful sentiment, there are some who then express a negative speculation, even when the hopeful sentiment was qualified with the acknowledgement that we don't know anything for sure. That feels like an attempt to squelch hope and I don't think that's helpful or healthy.

Its really not clear to me. The activation section is below the PCR section. Activated cells are not noted in connection with the PCR yet they are in other sections. Why point out that cells are activated on one section and not another? Did they use the primary cells for the PCR? Or did they just used the PBMC's as stated? What are primary cells? Does that denote something in particular?

They used PBMC's for the PCR; in the activation section they talk about splitting up the cells into their types - but they didn't do that for the PCR; they simply refer to PBMC's.

Note that it says the cells were either stored as unactivated cells for further culture or resuspended in Trizol for DNA and RNA extraction; ie one set of cells was cultured; the other set of cells was not - the uncultured cells were used for DNA and RNA extraction. I don't know how else to read that.

Yes, it seems to me that Gerwyn or someone else has patiently explained or countered every item, then someone has doggedly circled round the block and brought it up again and again. I don't know if that means they didn't understand the explanation, or they just didn't like the inference.

And FWIW, I don't need anyone to keep me from getting "too hopeful" (whatever that is) or to keep my feet on the ground. No need to save me from myself. I feel insulted and talked down to if anyone is posting negative speculation on XMRV just to keep me from getting too excited about it. Like Adam, the rumors and speculations I've seen here have been predominately negative on XMRV. Of course, if there is some real science that disproves WPIs preliminary study, bring it on. None has appeared so far.

And to describe any of Dr Mikovits's statements, at least those I'm aware of, as "lamblasting" anyone is hyperbole itself. Dr Vernon's speculation, if what Cort says of her is true, that purported flaws in WPIs study might somehow lower the interest of the rest of the scientific community in XMRV just isn't supportable by the facts. There are studies on XMRV going on all over the world. Governments, private researchers and BigPharma are all interested, no matter whether the criticisms of WPI are born out or proven to be more smoke.

What I am seeing is that whenever someone expresses a hopeful sentiment, there are some who then express a negative speculation, even when the hopeful sentiment was qualified with the acknowledgement that we don't know anything for sure. That feels like an attempt to squelch hope and I don't think that's helpful or healthy.

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i think that dr m was merely expressing her feelings when proffessional scientists seemed incapable of following relatively simple instructions

Yes, it seems to me that Gerwyn or someone else has patiently explained or countered every item, then someone has doggedly circled round the block and brought it up again and again. I don't know if that means they didn't understand the explanation, or they just didn't like the inference.

And FWIW, I don't need anyone to keep me from getting "too hopeful" (whatever that is) or to keep my feet on the ground. No need to save me from myself. I feel insulted and talked down to if anyone is posting negative speculation on XMRV just to keep me from getting too excited about it. Like Adam, the rumors and speculations I've seen here have been predominately negative on XMRV. Of course, if there is some real science that disproves WPIs preliminary study, bring it on. None has appeared so far.

And to describe any of Dr Mikovits's statements, at least those I'm aware of, as "lamblasting" anyone is hyperbole itself. Dr Vernon's speculation, if what Cort says of her is true, that purported flaws in WPIs study might somehow lower the interest of the rest of the scientific community in XMRV just isn't supportable by the facts. There are studies on XMRV going on all over the world. Governments, private researchers and BigPharma are all interested, no matter whether the criticisms of WPI are born out or proven to be more smoke.

What I am seeing is that whenever someone expresses a hopeful sentiment, there are some who then express a negative speculation, even when the hopeful sentiment was qualified with the acknowledgement that we don't know anything for sure. That feels like an attempt to squelch hope and I don't think that's helpful or healthy.

Gerwyn! I was hoping that you would see this (if not have not already investigated).

As for me, one of the areas of NOTICEABLE (to ME, anyway...if not to anyone else!) improvement is with Memory/cognitive. AND, PEM does NOT last as long (and is NOT as severe).
I continue to have many other problems (chronic reactivated shingles on BOTH sides...BUT they are quite manageable now!, hsv1, high EBV, CMV titres, etc.). I was instructed the other day to begin equilibrant NOW (newer version of Oxymatrine), in addition to Antiviral...to discontinue (for the time being) Tagamet. jackie

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I had Shingle when I was 9, and have had chicken pox more than 14 times. My health deteriorated and I was sick all the time after shingles. I appear to have been a normal child before that. (Although I did have pneumonia at 7.) It was after shingle I had the endless sore throats, swollen glands, headache, fatigue, and no apparent explanation. Can you tell me more about the chicken pox virus in connection with CFS, or where to look for more information?

I've been able to gradually improve my physical fitness and other bodily processes, but my brain is still in a fog. I really want my brain back!

Hi Noddyboddy! The best I can do for you is send you to where I've looked for info - which is one of Dr. Chia's sites..."EVMED research". Lots of info re Enteroviruses (coxsackie/echos etc.) and also VZV (cause of Shingles)....and his theories about their connection to me/cfs.

Interesting, although I was a very "sickly" child (pneumonia before I was 6 wks. old)...I didn't get Chickenpox until much older than normal - 16...and my first shingles about 4 years ago - about the time I started antivirals. I certainly sympathize with what YOU went through as a child!

I wish I knew how to give links or better yet, post the entire site here for informational purposes...but I haven't learned how! (so much for my "improved" mem/cog!)

Seriously, I credit my long term use of antivirals with an improvement (at times dramatic) in my Brain fog!...I only wish my body would catch up (and no more chronic shingles would also be nice!)

Thank-you to those who have answered my request for input on whether you want to hear more about HERVs, alternative hypothesis for XMRV, etc. What I gather from the responses is that it is split, some are interested and some do not think it is helpful. Kim mentioned the CFS population is strong enough to deal with the eventual outcomes if XMRV fails, although others disagreed. I sense in the conflict over just talking about possible problems with the XMRV studies, that the CFS population is not uniformly resilient. And also several have requested to separate speculation from fact more clearly. That is a good idea I think for everyone.

I need to take a rest from all this for awhile, so will probably not post as much the next few days. However, I want to reply to Gerwyn's repeated statements that there is no in vitro evidence for the points I made. There is good evidence, but I have not always provided quotes, expecting people would google search the issues I raise, but probably many are too tired for that. So I will try to look up these items and give quotes or references more often. Actually, I did give 'in vitro' evidence for the emerging hypthesis that HERVs are involved in disease, including work by Huber for HERV K18 in CFS, and a new study in MS showing differential HERV W expression between MS tissue samples and controls. And there is plenty of 'in vitro' evidence of HERV cross-reactivity, including with MuLV, in the 'Rumor Viruses' article I included. That is a long .pdf article and probably I need to hunt down some specific quotes. Someone else is welcome also to work through that article and find the evidence, it is there, over 500 study references!

Gerwyn, you stated that you are a qualified microbiologist. Would you please clarify what that means? I have some idea in the US, but maybe that should be more clear to everyone here given the level of analysis you provide. For example, here in the US that could mean several things, from a technician with a bachelor's degree running culture machines and operating a microscope in a hospital lab, to a PhD researcher in a laboratory conducting original studies. And probably several steps in between. And a second question, do you have formal training in virology beyond the introductory level? (some may not realize that virology is not necessarily part of microbiology, at least in the US, a distinct specialty) Thanks.